The primary function of the epidermis is to produce the protective, water-impermeable stratum corneum that permits terrestrial life. The barrier function is provided by a lipid mixture that originates from specialized organelles called lamellar granules (LGs) within the viable portion of the epidermis. The most prevalent lipids in the stratum corneum are ceramides, which are derived from precursor glucosylceramides extruded into the intercellular space at the junction of the viable epidermis and the stratum corneum. Lysosomal enzymes, which are likely packaged into LGs, are also extruded into the intercellular space and are responsible for the extensive lipid remodeling required to generate the barrier lipid. Little is known about the mechanisms and regulation of LG synthesis. With the knowledge that lipids are essential to both the structure and the primary function of skin, the long-term objectives of this application are first, to elucidate the mechanisms involved in lipid transport and metabolism in keratinocytes. Secondly to determine the relationship between epidermal differentiation and lipid metabolism. With these long-term objectives, the focus of this application is on the regulation of essential events involved in LG synthesis, intracellular trafficking, and extrusion. The application proposes to use keratinocyte cultures as an epidermal model. Biochemical, cell biological, and molecular genetic techniques will be used in this systems The investigators will determine: 1) the mechanisms by which CGT, the enzyme responsible for glucosylceramide synthesis, is regulated; 2) the functional role of CGT in LG assembly and epidermal differentiation and 3) the nature of the LG compartment with respect to defined intracellular compartments and the mechanisms underlying the exocytosis of LG contents. Basic knowledge about epidermal lipid metabolism has important biomedical applications. Epidermal differentiation and stratum corneum barrier function are disturbed in a variety of skin diseases, but for many of these, the underlying mechanism remains unknown. Either secondary changes, or as yet undiscovered primary genetic defects, in many of the cellular processes associated with lipid metabolism are likely to be responsible for this disruption of epidermal function. Understanding normal epidermal, lipid metabolism is vital to understand the role that disturbances in these processes may play in cutaneous disease and to devise rational therapies. Ultimately, these studies will contribute to fundamental knowledge about the regulation of epidermal differentiation at the molecular level.